Summary table with the time needed to perform each task of the TCR-SMART protocol

Task	Timing	Day
Identification of TCR sequences in single-cell TCR-seq data	3 h	Before you begin
qPCR primer design	10 min (per primer pair)	Before you begin
Isolation of PMBCs	1.5 h	Day 1
Magnetic sorting of CD14+ monocytes	70 min	Day 1
Irradiation of CD14+ cells and antigen loading	75 min hands-on + 4 h of incubation	Day 1
Sorting of memory CD4+ T cells	1.5 h + FACS sorting time	Day 1
CD4+ T cells and antigen-loaded monocytes co-culture	1 h + 6 days of incubation	Day 1
Isolation of antigen-specific CD4+ T cells	85 min + FACS sorting time	Day 7
Identification of defined CD4+ T cell clones by TCR-SMART	4 h + 2 h per qPCR plate	Day 7–8